AMP-forming acetyl-CoA synthetase (AMP-ACS) is the principal enzyme for the conversion of acetate to acetyl-CoA, an essential intermediate at the junction of anabolic and catabolic pathways. Although progress has been made in understanding the physiological roles of AMP-ACS, its biochemistry has received little attention. AMP-ACS is a member of a diverse superfamily of acyl-adenylate-forming enzymes that includes acyl-CoA ligases, enzymes that mediate the synthesis of peptide and polyketide secondary metabolites, firefly luciferase, and ( aminoadipate reductase. Although structures have been solved for several members of this family, AMP-ACS is too distantly related to allow molecular modeling. The recent structure of the Salmonella enterica AMP-ACS now allows predictions to be made regarding the active site and residues important for substrate binding and catalysis. The two specific aims of the proposed study are (1) to identify residues in the acetate binding pocket and (2) to identify residues important for ATP binding. Analysis of the S. enterica structure and sequence alignment between AMP-ACSs and the very closely related to the propionyl-CoA synthetases, which utilize propionate as substrate instead of acetate, and human SA and MACS 1, which utilize isobutyrate and octanoate, respectively, allowed candidate residues that may be important in binding acetate or ATP to be identified. Alteration of these residues by site-directed mutagenesis followed by kinetic and biochemical characterization of the resulting variants will delineate their role in substrate binding.